All-in-One: Multifunctional Hydrogel Accelerates Oxidative Diabetic Wound Healing through Timed-Release of Exosome and Fibroblast Growth Factor.

The treatment of diabetic wounds remains a major challenge in clinical practice, with chronic wounds characterized by multiple drug-resistant bacterial infections, angiopathy, and oxidative damage to the microenvironment. Herein, a novel in situ injectable HA@MnO2 /FGF-2/Exos hydrogel is introduced for improving diabetic wound healing. Through a simple local injection, this hydrogel is able to form a protective barrier covering the wound, providing rapid hemostasis and long-term antibacterial protection. The MnO2 /ε-PL nanosheet is able to catalyze the excess H2 O2 produced in the wound, converting it to O2 , thus not only eliminating the harmful effects of H2 O2 but also providing O2 for wound healing. Moreover, the release of M2-derived Exosomes (M2 Exos) and FGF-2 growth factor stimulates angiogenesis and epithelization, respectively. These in vivo and in vitro results demonstrate accelerated healing of diabetic wounds with the use of the HA@MnO2 /FGF-2/Exos hydrogel, presenting a viable strategy for chronic diabetic wound repair.

Metformin alleviates bone loss in ovariectomized mice through inhibition of autophagy of osteoclast precursors mediated by E2F1.

BACKGROUND: Postmenopausal bone loss, mainly caused by excessive bone resorption mediated by osteoclasts, has become a global public health burden. Metformin, a hypoglycemic drug, has been reported to have beneficial effects on maintaining bone health. However, the role and underlying mechanism of metformin in ovariectomized (OVX)-induced bone loss is still vague. RESULTS: In this study, we demonstrated for the first time that metformin administration alleviated bone loss in postmenopausal women and ovariectomized mice, based on reduced bone resorption markers, increased bone mineral density (BMD) and improvement of bone microstructure. Then, osteoclast precursors administered metformin in vitro and in vivo were collected to examine the differentiation potential and autophagical level. The mechanism was investigated by infection with lentivirus-mediated BNIP3 or E2F1 overexpression. We observed a dramatical inhibition of autophagosome synthesis and osteoclast formation and activity. Treatment with RAPA, an autophagy activator, abrogated the metformin-mediated autophagy downregulation and inhibition of osteoclastogenesis. Additionally, overexpression of E2F1 demonstrated that reduction of OVX-upregulated autophagy mediated by metformin was E2F1 dependent. Mechanistically, metformin-mediated downregulation of E2F1 in ovariectomized mice could downregulate BECN1 and BNIP3 levels, which subsequently perturbed the binding of BECN1 to BCL2. Furthermore, the disconnect between BECN1 and BCL2 was shown by BNIP3 overexpression. CONCLUSION: In summary, we demonstrated the effect and underlying mechanism of metformin on OVX-induced bone loss, which could be, at least in part, ascribed to its role in downregulating autophagy during osteoclastogenesis via E2F1-dependent BECN1 and BCL2 downregulation, suggesting that metformin or E2F1 inhibitor is a potential agent against postmenopausal bone loss. Video abstract.

LncRNA KCNQ1OT1 accelerates fracture healing via modulating miR-701-3p/FGFR3 axis.

Emerging evidence highlights the role of the long noncoding RNA (lncRNA) KCNQ1OT1 in fracture healing. Osteoblast proliferation, migration, and survival are pivotal during this process. In this study, we aimed to improve our understanding of the regulatory role of lncRNA KCNQ1OT1 during osteoblast proliferation, migration, and survival. We searched the gene expression omnibus databases and LncBase Experimental V.2 to identify key microRNAs (miRNAs) targets of KCNQ1OT1. MiR-701-3p was selected as a differentially expressed miRNA and RNA immunoprecipitation assays were performed to verify its interaction with KCNQ1OT1. Fibroblast growth factor receptor 3 (FGFR3) was also identified as a target of miR-701-3p. We further identified KCNQ1OT1 as a competing endogenous RNA of miR-701-3p that could influence osteoblast proliferation, migration, and apoptosis in vitro and in vivo. Taken together, our results indicate that the KCNQ1OT1/miR-701-3p/FGFR3 axis is an important regulator of osteoblast proliferation, migration, and apoptosis, and provide a new therapeutic avenue for fracture healing.

Exosomes derived from pioglitazone-pretreated MSCs accelerate diabetic wound healing through enhancing angiogenesis.

BACKGROUND: Enhanced angiogenesis can promote diabetic wound healing. Mesenchymal stem cells (MSCs)-derived exosomes, which are cell-free therapeutics, are promising candidates for the treatment of diabetic wound healing. The present study aimed to investigate the effect of exosomes derived from MSCs pretreated with pioglitazone (PGZ-Exos) on diabetic wound healing. RESULTS: We isolated PGZ-Exos from the supernatants of pioglitazone-treated BMSCs and found that PGZ-Exos significantly promote the cell viability and proliferation of Human Umbilical Vein Vascular Endothelial Cells (HUVECs) injured by high glucose (HG). PGZ-Exos enhanced the biological functions of HUVECs, including migration, tube formation, wound repair and VEGF expression in vitro. In addition, PGZ-Exos promoted the protein expression of p-AKT, p-PI3K and p-eNOS and suppressed that of PTEN. LY294002 inhibited the biological function of HUVECs through inhibition of the PI3K/AKT/eNOS pathway. In vivo modeling in diabetic rat wounds showed that pioglitazone pretreatment enhanced the therapeutic efficacy of MSCs-derived exosomes and accelerated diabetic wound healing via enhanced angiogenesis. In addition, PGZ-Exos promoted collagen deposition, ECM remodeling and VEGF and CD31 expression, indicating adequate angiogenesis in diabetic wound healing. CONCLUSIONS: PGZ-Exos accelerated diabetic wound healing by promoting the angiogenic function of HUVECs through activation of the PI3K/AKT/eNOS pathway. This offers a promising novel cell-free therapy for treating diabetic wound healing.

Methyltransferase-like 3-mediated N6-methyladenosine modification of miR-7212-5p drives osteoblast differentiation and fracture healing.

N6-methyladenosine (m6A) modification has been reported in various diseases and implicated in increasing numbers of biological processes. However, previous studies have not focused on the role of m6A modification in fracture healing. Here, we demonstrated that m6A modifications are decreased during fracture healing and that methyltransferase-like 3 (METTL3) is the main factor involved in the abnormal changes in m6A modifications. Down-regulation of METTL3 promotes osteogenic processes both in vitro and in vivo, and this effect is recapitulated by the suppression of miR-7212-5p maturation. Further studies have shown that miR-7212-5p inhibits osteoblast differentiation in MC3T3-E1 cells by targeting FGFR3. The present study demonstrated an important role of the METTL3/miR-7212-5p/FGFR3 axis and provided new insights on m6A modification in fracture healing.

IL-10 induces MC3T3-E1 cells differentiation towards osteoblastic fate in murine model.

Interleukin-10 (IL-10) displays well-documented anti-inflammatory effects, but its effects on osteoblast differentiation have not been investigated. In this study, we found IL-10 negatively regulates microRNA-7025-5p (miR-7025-5p), the down-regulation of which enhances osteoblast differentiation. Furthermore, through luciferase reporter assays, we found evidence that insulin-like growth factor 1 receptor (IGF1R) is a miR-7025-5p target gene that positively regulates osteoblast differentiation. In vivo studies indicated that the pre-injection of IL-10 leads to increased bone formation, while agomiR-7025-5p injection delays fracture healing. Taken together, these results indicate that IL-10 induces osteoblast differentiation via regulation of the miR-7025-5p/IGF1R axis. IL-10 therefore represents a promising therapeutic strategy to promote fracture healing.

Circulating Exosomal miR-20b-5p Inhibition Restores Wnt9b Signaling and Reverses Diabetes-Associated Impaired Wound Healing.

At present, developing therapeutic strategies to improve wound healing in individuals with diabetes remains challenging. Exosomes represent a promising nanomaterial from which microRNAs (miRNAs) can be isolated. These miRNAs have the potential to exert therapeutic effects, and thus, determining the potential therapeutic contributions of specific miRNAs circulating in exosomes is of great importance. In the present study, circulating exosomal miRNAs are identified in diabetic patients and assessed for their roles in the context of diabetic wound healing. A significant upregulation of miR-20b-5p is observed in exosomes isolated from patients with type 2 diabetes mellitus (T2DM), and this miRNA is able to suppress human umbilical vein endothelial cell angiogenesis via regulation of Wnt9b/ß-catenin signaling. It is found that the application of either miR-20b-5p or diabetic exosomes to wound sites is sufficient to slow wound healing and angiogenesis. In diabetic mice, it is found that knocking out miR-20b-5p significantly enhances wound healing and promotes wound angiogenesis. Together, these findings thus provide strong evidence that miR-20b-5p is highly enriched in exosomes from patients with T2DM and can be transferred to cells of the vascular endothelium, where it targets Wnt9b signaling to negatively regulate cell functionality and angiogenesis.

Comparison of different methods for the isolation and purification of rat nucleus pulposus-derived mesenchymal stem cells.

PURPOSE: Recently, nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have been identified and have shown good prospects for the repair of degenerative intervertebral discs. However, there is no consensus about the methods for the isolation and purification of NPMSCs. Therefore, a reliable and efficient isolation and purification method is potentially needed. We aimed to compare different methods and to identify an optimal method for isolating and purifying NPMSCs. METHODS: NPMSCs were isolated and purified using two common methods (a low-density culture (LD) method and a mesenchymal stem cell complete medium culture (MSC-CM) method) and two novel methods (a cloning cylinder (CC) method and a combination of the CC and MSC-CM methods (MSC-CM+CC)). The morphology, MSC-specific surface markers (CD44, CD73, CD90, CD105, CD34 and HLA-DR), multiple-lineage differentiation potential, colony formation ability, and stemness gene (Oct4, Nanog, and Sox2) expression were evaluated and compared. RESULTS: NPMSCs isolated from nucleus pulposus (NP) tissues via the four methods met the criteria stated by the International Society of Cell Therapy (ISCT) for MSCs, including adherent growth ability, MSC-specific surface antigen expression, and multi-lineage differentiation potential. In particular, the MSC-CM+CC method yielded a relatively higher quality of NPMSCs in terms of cell surface markers, multiple-lineage differentiation potential, colony formation ability, and stemness gene expression. CONCLUSIONS: Our results indicated that NPMSCs can be obtained via all four methods and that the MSC-CM+CC method is more reliable and efficient than the other three methods. The findings from this study provide an alternative option for isolating and purifying NPMSCs.

Saliva exosomes-derived UBE2O mRNA promotes angiogenesis in cutaneous wounds by targeting SMAD6.

BACKGROUND: Enhancing angiogenesis is critical for accelerating wound healing. Application of different types of exosomes (Exos) to promote angiogenesis represents a novel strategy for enhanced wound repair. Saliva is known to accelerate wound healing, but the underlying mechanisms remain unclear. RESULTS: Our results have demonstrated that saliva-derived exosomes (saliva-Exos) induce human umbilical vein endothelial cells (HUVEC) proliferation, migration, and angiogenesis in vitro, and promote cutaneous wound healing in vivo. Further experiments documented that Ubiquitin-conjugating enzyme E2O (UBE2O) is one of the main mRNAs of saliva-Exos, and activation of UBE2O has effects similar to those of saliva-Exos, both in vitro and in vivo. Mechanistically, UBE2O decreases the level of SMAD family member 6 (SMAD6), thereby activating bone morphogenetic protein 2 (BMP2), which, in turn, induces angiogenesis. CONCLUSIONS: The present work suggests that administration of saliva-Exos and UBE2O represents a promising strategy for enhancing wound healing through promotion of angiogenesis.

M2 Macrophagy-derived exosomal miRNA-5106 induces bone mesenchymal stem cells towards osteoblastic fate by targeting salt-inducible kinase 2 and 3.

BACKGROUND: Osteoblast differentiation is a vital process for fracture healing, and exosomes are nanosized membrane vesicles that can deliver therapeutic drugs easily and safely. Macrophages participate in the regulation of various biological processes in vivo, and macrophage-derived exosomes (MD-Exos) have recently been a topic of increasing research interest. However, few study has explored the link between MD-Exos and osteoblast differentiation. Herein, we sought to identify miRNAs differentially expressed between M1 and M2 macrophage-derived exosomes, and to evaluate their roles in the context of osteoblast differentiation. RESULTS: We found that microRNA-5106 (miR-5106) was significantly overexpressed in M2 macrophage-derived exosomes (M2D-Exos), while its expression was decreased in M1 macrophage-derived exosomes (M1D-Exos), and we found that this exosomal miRNA can induce bone mesenchymal stem cell (BMSC) osteogenic differentiation via directly targeting the Salt-inducible kinase 2 and 3 (SIK2 and SIK3) genes. In addition, the local injection of both a miR-5106 agonist or M2D-Exos to fracture sites was sufficient to accelerate healing in vivo. CONCLUSIONS: Our study demonstrates that miR-5106 is highly enriched in M2D-Exos, and that it can be transferred to BMSCs wherein it targets SIK2 and SIK3 genes to promote osteoblast differentiation.

High-heeled-related alterations in the static sagittal profile of the spino-pelvic structure in young women.

PURPOSE: Women wearing high-heeled shoes have been considered to be more characterizing beauty, self-assurance and elegance. However, while maintaining the body on this type of support base, women with increased heel height often complain that wearing high-heeled shoes causes them to experience low back pain. The aim of the present study was to morphologically assess the effect of high-heel use on the static sagittal profile of the spino-pelvic structure. METHODS: A total of 21 Chinese girls were recruited in this study, with informed written consent. For each participant, standing left lateral radiographs, including that of the spine and pelvis, were obtained in a standardized standing position under barefoot and high-heel use conditions. The radiographic assessments were performed to detect the changes in the spino-pelvic profile under barefoot and high-heel use conditions. RESULTS: The average lumbar lordosis (LL) was 54.3 ± 6.4º under the barefoot condition and increased to 65.2 ± 5.1º after high-heel use (P < 0.001), with a significant increase in the disc L5/S1 and disc L4/L5 tilt angles. Of the 21 participants, 15 (71.43 %) had an increased kyphosis value for thoracic kyphosis, and 6 (28.57 %) had a decreased value after high-heel use, with a significant increased mean kyphosis value of 3.4 ± 1.5º overall (P < 0.001). The sagittal vertical axis (SVA) was always positive and was worse after high-heel use (P = 0.012): 11.5 ± 8.7 mm under the barefoot condition and 29.8 ± 8.5 mm under the high-heel use condition. Bivariate correlation analysis showed that both ΔLL and ΔSVA were positively associated with the heel height of the shoes and were inversely associated with the age of the participants. Receiver operator characteristic analysis showed that a heel height >45.5 mm was strongly predictive of the loss of static sagittal balance of the spine during high-heel use (sensitivity 87.5 %, specificity 62.5 %, area under the curve: 0.773; P = 0.026). CONCLUSIONS: The present study revealed that wearing high-heeled shoes can lead to increased LL and an uneconomic body position. This finding may help explain why some women complain that wearing high-heeled shoes causes them to experience low back pain.

Regulation of metabolic microenvironment with a nanocomposite hydrogel for improved bone fracture healing.

Bone nonunion poses an urgent clinical challenge that needs to be addressed. Recent studies have revealed that the metabolic microenvironment plays a vital role in fracture healing. Macrophages and bone marrow-derived mesenchymal stromal cells (BMSCs) are important targets for therapeutic interventions in bone fractures. Itaconate is a TCA cycle metabolite that has emerged as a potent macrophage immunomodulator that limits the inflammatory response. During osteogenic differentiation, BMSCs tend to undergo aerobic glycolysis and metabolize glucose to lactate. Copper ion (Cu2+) is an essential trace element that participates in glucose metabolism and may stimulate glycolysis in BMSCs and promote osteogenesis. In this study, we develop a 4-octyl itaconate (4-OI)@Cu@Gel nanocomposite hydrogel that can effectively deliver and release 4-OI and Cu2+ to modulate the metabolic microenvironment and improve the functions of cells involved in the fracture healing process. The findings reveal that burst release of 4-OI reduces the inflammatory response, promotes M2 macrophage polarization, and alleviates oxidative stress, while sustained release of Cu2+ stimulates BMSC glycolysis and osteogenic differentiation and enhances endothelial cell angiogenesis. Consequently, the 4-OI@Cu@Gel system achieves rapid fracture healing in mice. Thus, this study proposes a promising regenerative strategy to expedite bone fracture healing through metabolic reprogramming of macrophages and BMSCs.

Bone-targeting engineered small extracellular vesicles carrying anti-miR-6359-CGGGAGC prevent valproic acid-induced bone loss.

The clinical role and underlying mechanisms of valproic acid (VPA) on bone homeostasis remain controversial. Herein, we confirmed that VPA treatment was associated with decreased bone mass and bone mineral density (BMD) in both patients and mice. This effect was attributed to VPA-induced elevation in osteoclast formation and activity. Through RNA-sequencing, we observed a significant rise in precursor miR-6359 expression in VPA-treated osteoclast precursors in vitro, and further, a marked upregulation of mature miR-6359 (miR-6359) in vivo was demonstrated using quantitative real-time PCR (qRT-PCR) and miR-6359 fluorescent in situ hybridization (miR-6359-FISH). Specifically, the miR-6359 was predominantly increased in osteoclast precursors and macrophages but not in neutrophils, T lymphocytes, monocytes and bone marrow-derived mesenchymal stem cells (BMSCs) following VPA stimulation, which influenced osteoclast differentiation and bone-resorptive activity. Additionally, VPA-induced miR-6359 enrichment in osteoclast precursors enhanced reactive oxygen species (ROS) production by silencing the SIRT3 protein expression, followed by activation of the MAPK signaling pathway, which enhanced osteoclast formation and activity, thereby accelerating bone loss. Currently, there are no medications that can effectively treat VPA-induced bone loss. Therefore, we constructed engineered small extracellular vesicles (E-sEVs) targeting osteoclast precursors in bone and naturally carrying anti-miR-6359 by introducing of EXOmotif (CGGGAGC) in the 3'-end of the anti-miR-6359 sequence. We confirmed that the E-sEVs exhibited decent bone/osteoclast precursor targeting and exerted protective therapeutic effects on VPA-induced bone loss, but not on ovariectomy (OVX) and glucocorticoid-induced osteoporotic models, deepening our understanding of the underlying mechanism and treatment strategies for VPA-induced bone loss.

Hallmarks of peripheral nerve function in bone regeneration.

Skeletal tissue is highly innervated. Although different types of nerves have been recently identified in the bone, the crosstalk between bone and nerves remains unclear. In this review, we outline the role of the peripheral nervous system (PNS) in bone regeneration following injury. We first introduce the conserved role of nerves in tissue regeneration in species ranging from amphibians to mammals. We then present the distribution of the PNS in the skeletal system under physiological conditions, fractures, or regeneration. Furthermore, we summarize the ways in which the PNS communicates with bone-lineage cells, the vasculature, and immune cells in the bone microenvironment. Based on this comprehensive and timely review, we conclude that the PNS regulates bone regeneration through neuropeptides or neurotransmitters and cells in the peripheral nerves. An in-depth understanding of the roles of peripheral nerves in bone regeneration will inform the development of new strategies based on bone-nerve crosstalk in promoting bone repair and regeneration.

Recent advances in GelMA hydrogel transplantation for musculoskeletal disorders and related disease treatment.

Increasing data reveals that gelatin that has been methacrylated is involved in a variety of physiologic processes that are important for therapeutic interventions. Gelatin methacryloyl (GelMA) hydrogel is a highly attractive hydrogels-based bioink because of its good biocompatibility, low cost, and photo-cross-linking structure that is useful for cell survivability and cell monitoring. Methacrylated gelatin (GelMA) has established itself as a typical hydrogel composition with extensive biomedical applications. Recent advances in GelMA have focused on integrating them with bioactive and functional nanomaterials, with the goal of improving GelMA's physical, chemical, and biological properties. GelMA's ability to modify characteristics due to the synthesis technique also makes it a good choice for soft and hard tissues. GelMA has been established to become an independent or supplementary technology for musculoskeletal problems. Here, we systematically review mechanism-of-action, therapeutic uses, and challenges and future direction of GelMA in musculoskeletal disorders. We give an overview of GelMA nanocomposite for different applications in musculoskeletal disorders, such as osteoarthritis, intervertebral disc degeneration, bone regeneration, tendon disorders and so on.

Melatonin Promotes BMSCs Osteoblastic Differentiation and Relieves Inflammation by Suppressing the NF-κB Pathways.

Bone mesenchymal stem cells (BMSCs) play an important role in maintaining the dynamic balance of bone metabolism. Recent studies have reported that a decrease in the osteogenic function of MSCs is strongly associated with osteoporosis. Melatonin is a neuroendocrine hormone produced in the pineal gland and is essential in the physiological regulation. This study is aimed at exploring the effect of melatonin on MSCs osteoblastic differentiation and elucidate the underlying mechanisms. We isolated BMSCs from rat bone marrow and demonstrated that melatonin improved osteogenic differentiation of BMSCs by the alizarin red staining and ALP staining. We then showed that melatonin enhanced osteogenic gene expression in BMSCs, including ALP, Col 1, OCN, OPN, and RUNX2. We further revealed that melatonin inhibited the inflammatory response of BMSCs by suppressing the NF-κB signaling pathways. In light of this, we found that the NF-κB pathway-specific activator TNF-α activated the NF-κB pathway, inhibited osteogenic differentiation, and induced inflammatory response in BMSCs. Melatonin was found to reverse the inhibitory effect of TNF-α on osteogenic differentiation and inflammation in BMSCs. Taken together, these findings indicated that melatonin may have therapeutic potential to be used for the treatment of osteoporosis.

A Whole-Course-Repair System Based on Neurogenesis-Angiogenesis Crosstalk and Macrophage Reprogramming Promotes Diabetic Wound Healing.

Diabetic wound (DW) therapy is currently a big challenge in medicine and strategies to enhance neurogenesis and angiogenesis have appeared to be a promising direction. However, the current treatments have failed to coordinate neurogenesis and angiogenesis simultaneously, leading to an increased disability rate caused by DWs. Herein, a whole-course-repair system is introduced by a hydrogel to concurrently achieve a mutually supportive cycle of neurogenesis-angiogenesis under a favorable immune-microenvironment. This hydrogel can first be one-step packaged in a syringe for later in situ local injections to cover wounds long-termly for accelerated wound healing via the synergistic effect of magnesium ions (Mg2+ ) and engineered small extracellular vesicles (sEVs). The self-healing and bio-adhesive properties of the hydrogel make it an ideal physical barrier for DWs. At the inflammation stage, the formulation can recruit bone marrow-derived mesenchymal stem cells to the wound sites and stimulate them toward neurogenic differentiation, while providing a favorable immune microenvironment via macrophage reprogramming. At the proliferation stage of wound repair, robust angiogenesis occurs by the synergistic effect of the newly differentiated neural cells and the released Mg2+ , allowing a regenerative neurogenesis-angiogenesis cycle to take place at the wound site. This whole-course-repair system provides a novel platform for combined DW therapy.